1. Field of the Invention
The present invention is in the fields of molecular biology, cell biology, and genetics. The invention is directed generally to mutating genes in cells in vitro and in multi-cellular organisms. The invention encompasses methods for mutating genes in cells using a combination of mutagens, wherein at least one mutagen is a polynucleotide that acts as an insertional mutagen. Such methods are used to achieve mutation of a single gene to achieve a desired phenotype as well as mutation of multi-cellular organism. The invention is also directed to methods of identifying one or more mutated genes, made by the methods of the invention, in cells and in multi-cellular organisms, by means of a tagging property provided by the insertional mutagen(s). The insertional mutagen thus allows identification of one or more genes that are mutated by insertion of the insertional mutagen.
The invention is also directed to cells and multi-cellular organisms created by the methods of the invention and uses of the cells and multicellular organisms. The invention is also directed to libraries of cells created by the methods of the invention and uses of the libraries.
2. Background
Mutagenesis has been used to identify the function of a large and growing number of genes. Mutation of one or more genes in a multi-cellular organism or cell allows the artisan to study the mutant organism or cell and compare it to the non-mutagenized (which may be wildtype) parent organism or cell. By identifying phenotypes associated with the mutant organism or cell, the function of the mutated gene(s) can be ascertained. Furthermore, mutagenesis provides a means for altering the genetic make up of a cell or multi-cellular organism to obtain a desired result. For example, it may be desirable to create a physiological disorder in a eukaryotic organism by mutating one more genes and then to identify one or more of the relevant genes. Thus, mutations that have a desired use (e.g., for commercial production of proteins, foodstuffs, or pharmaceuticals, or for production of transgenic animals as models of certain diseases) can be identified and selected. The possibilities for use of this technology, whether in vitro, ex vivo, or in vivo, are well known in the art.
Identification of novel genes and characterization of their function using mutagenesis has also been shown to be productive in identifying new drugs and drug targets. Creating in vitro cellular models that exhibit phenotypes that are clinically relevant provides a valuable substrate for target identification and screening for compounds that modulate not only the phenotype but also the target(s) that controls the phenotype. Modulation of such a target can provide information that validates the target as important for therapeutic intervention in a clinical disorder when such modulation of the target serves to modulate a clinically relevant phenotype.
Animal models exhibiting clinically relevant phenotypes are also valuable for drug discovery and development and for drug target identification. For example, mutation of somatic or germ cells facilitates the production of genetically modified offspring or cloned animals having a phenotype of interest. Such animals have a number of uses, for example as models of physiological disorders (e.g., of human genetic diseases) that are useful for screening the efficacy of candidate therapeutic compounds or compositions for treating or preventing such physiological disorders. Furthermore, identifying the gene(s) responsible for the phenotype provides potential drug targets for modulating the phenotype and, when the phenotype is clinically relevant, for therapeutic intervention. In addition, the manipulation of the genetic makeup of organisms and the identification of new genes have important uses in agriculture, for example in the development of new strains of animals and plants having higher nutritional value or increased resistance to environmental stresses (such as heat, drought, or pests) relative to their wildtype or non-mutant counterparts.
Since most eukaryotic cells are diploid, two copies of most genes are present in each cell. As a consequence, homozygous mutation is usually required to produce a desired phenotype, since mutating one copy of a gene may not produce a sufficient change in the level of gene expression or activity of the gene product from that in the non-mutated or wildtype cell or multicellular organism, and since the remaining wildtype copy would still be expressed at sufficient levels to produce a functional gene product. Thus, to create a desired change in the level of gene expression and/or function in a cell or multicellular organism, at least two mutations, one in each copy of the gene, are required in the same cell.
In other instances, mutation in multiple genes may be required to produce a desired phenotype. In some instances, a mutation in one copy of a gene may affect the expression levels of the gene but not the activity of the gene product to a desired extent, so that the desired physiological effects on the cell or multi-cellular organism is not achieved. However, a mutation in a second gene, even in only one copy of that second gene, can reduce gene expression levels of the second gene to produce a cumulative phenotypic effect in combination with the first mutation, if the expression levels of both genes are sufficiently low. This effect can alter the function of a cell or multi-cellular organism. An example of this phenomenon is the synergy between blood clotting Factors VIII and IX. A mutation in either gene alone could result in levels that are severely reduced but with no effect on the clotting function. Severe reductions in the level of expression of both genes, however, can have a major impact. This principle can be extended to other instances where mutations in multiple (two, three, four, or more, for example) genes are required cumulatively to produce an effect on activity of a gene product or on another phenotype in a cell or multi-cellular organism. It should be noted that, in this instance, such genes may all be expressed in the same cell type and therefore, all of the required mutations occur in the same cell. However, the genes may normally be expressed in different cell types (for example, secreting the different gene products from the different cells). In this case, the gene products are expressed in different cells but still have a biochemical relationship such that one or more mutations in each gene is required to produce the desired phenotype.
Unfortunately, few methods exist for creating cultured cells that contain multiple gene mutations that produce, cumulatively, a desired phenotype. Such methods often are time-consuming and prone to error. In addition, it is often very difficult or impossible to identify the genes that have been mutated using such methods.
Further, methods for making homozygous mutations in cultured cells, where the mutated genes are not known in advance of mutation, are not known to currently exist. Still further, without a way to identify a homozygous mutation, the artisan cannot associate the phenotype with a given mutation. Currently, to associate a desired phenotype with a homozygous mutation in a cultured cell, the location, structure and/or function of the gene must be known to the artisan in advance. Hence, the methods of mutation known in the art are not suitable for homozygously mutating a cell to achieve a desired phenotype and identifying the gene(s) responsible for the phenotype. Nor are there methods suitable for making cells with multiple mutations that cumulatively produce a desired phenotype and identifying the genes responsible for the phenotype.
Several approaches for introducing mutations into eukaryotic genes are currently in use. Each has significant limitations.
One approach is homologous recombination to mutate the level of gene expression or activity of a gene product in a cell. 1: Montgomery et al., Cell. 1991 Feb. 22;64(4):693-702; 2: Riele et al. Nature. 1990 Dec. 13;348(6302):649-51; 3: Mansour et. al., Proc Natl Acad Sci USA. 1990 October;87(19):7688-92; 4: Koller et al., Proc Natl Acad Sci USA. 1989 November;86(22):8927-31; 5: Capecchi M R. Science. 1989 Jun. 16;244(4910):1288-92; 6: Zimmer A, Gruss P. Nature. 1989 Mar. 9;338(6211):150-3; 7: Joyner A L, Skarnes W C, Rossant J. Nature. 1989 Mar. 9;338(6211):153-6; 8: Thompson S, Clarke A R, Pow A M, Hooper M L, Melton D W. Cell. 1989 Jan. 27;56(2):313-21; 9: Doetschman T, Maeda N, Smithies O. Proc Natl Acad Sci USA. 1998 November;85(22):8583-7; 10: Doetschman T, Gregg R G, Maeda N, Hooper M L, Melton D W, Thompson S, Smithies O. Nature. 1987 Dec. 10-16;330(6148):576-8; 11: Thomas K R, Capecchi M R. Cell. 1987 Nov. 6;51(3):503-12.
Typically, this approach is taken in embryonic stem cells or embryonic germ cells, which are used to make transgenic animals carrying the mutation of interest. An important limitation of this approach is that the gene to be mutated must be known in advance of mutation, cloned and sequenced to ensure that the mutagenic vector used in homologous recombination contains the appropriate targeting sequences. Furthermore, the process is laborious and results in only one mutant copy of the gene of interest in the cell. Where a phenotype depends on homozygosity for expression, the heterozygous cell, therefore, cannot be used to screen for a change in a phenotype of interest unless additional work is carried out to eliminate the second copy of the gene by homologous recombination. This additional work is time consuming and expensive, and more importantly can only be done on genes that are known to the artisan in advance.
Such mutated heterozygous cells can be used to make transgenic animals. However, such animals will also be heterozygous and may not express a phenotype different from the wildtype or nonmutant animal. Further breeding of the animals to homozygosity is therefore required if one desires to analyze the phenotypic effect of the mutation. Such breeding is time consuming and expensive.
Another approach involves chemical mutagenesis of cells and/or organisms (see, e.g., Brown et al., Hum. Mol. Genet. 7:1627-1633 (1998); Chen et al., Nature Gen. 24:314-317 (2000); Munroe et al., Nature Gen. 24:318-321 (2000); Nolan et al., Nature Gen. 25:440-443 (2000); the disclosures of all of which are incorporated herein by reference in their entireties for teaching the use of ENU to generate mutations that result in detectable phenotypes in cells or animals). This approach relies upon the use of one or more chemical mutagens that are able to produce one or more mutations in the genome. As is the case for mutation by homologous recombination, however, chemical mutagenesis also typically results in mutagenesis of only a single copy of a given gene. Since in cases where homozygous mutation is required to achieve a desired phenotype, both copies of a given gene must be mutated before a desired phenotype can be achieved, cells or organisms that undergo a single round of chemical mutagenesis typically do not show a desired change in phenotype. Hence, these cells or organisms generally are not useful for achieving for a desired phenotype.
A further problem is that while chemical mutagenesis results in the mutation of one or more genes in a cell, there is no straightforward way to determine the mutated gene(s) responsible for the phenotype. This approach also fails to provide a method for making multiple mutations that cumulatively provide a desired phenotype that also permits the genes responsible for the phenotype to be easily identified.
As discussed above (for homologous recombination mutagenesis) mutated heterozygous cells prepared by chemical mutagenesis can be used to create transgenic animals. However, the animals will also be heterozygous and may not, therefore, manifest a change in a desired phenotype from the wildtype. Time-consuming and costly breeding of the animals to homozygosity is required. Even if a change in the desired phenotype is observed in the transgenic animals (even in homozygous transgenic animals), it is very difficult, if not impossible, to identify the mutated gene(s) responsible for the phenotype. Therefore, a large number of breedings must be carried out to clone the mutated gene by standard positional cloning methods. Hence, this process is slow, expensive, difficult to carry out on large numbers of mutant animals, and has a high failure rate. Thus, chemical mutagenesis fails to provide homozygous mutations in cultured cells (and hence, in transgenic animals produced from such cells) and fails to provide a simple way to identify the mutated gene(s) responsible for a phenotype in cultured cells or in multi-cellular organisms.
Another approach that has been used to mutate genes involves the use of insertional mutagens, such as gene trap vectors, to mutate genes (e.g., Amsterdam et al., Genes Dev. 13:2713-2724 (1999); von Melcher et al., Genes Dev. 6:919-927 (1992); Gogos et al., J. Virol. 71:1644-1650 (1997); Voss et al., Dev. Dyn. 212:171-180 (1998); Zambrowicz et al., Proc. Natl. Acad. Sci. USA 94:3789-3794 (1997); Friedrich et al., Genes Dev. 5:1513-1523 (1991); the disclosures of all of which are incorporated herein by reference in their entireties for teaching the use of gene traps as a mutagenesis technique). These vectors are typically inserted into the genome of a cell by non-homologous recombination. Upon insertion, these vectors are designed to disrupt transcription and/or translation of a gene. Unfortunately, gene trap vectors are inefficient mutagens and mutate only one copy of a given gene. As a result, homozygous mutations typically cannot be created in cell culture with such mutagens. In animals, the mutant animal must be bred to homozygosity of the mutant gene prior to phenotypic analysis. Since it is difficult and expensive to breed large numbers of animals to homozygosity, this approach has only been used on a relatively small number of genes to date.
This approach also fails to provide a method for making multiple mutations that cumulatively provide a desired phenotype and where the genes responsible for the phenotype can be identified. The probabilities of achieving, in a single cell, insertions in each of the genes required, is low and decreases with the number of genes required to be mutated in order to achieve the desired phenotype. Thus, gene traps fail to mutate multiple genes and fail to create homozygous mutations in cultured cells.
Stark et al. (Human Molecular Genetics 8:1925-1938 (1999)), in a review article on forward genetics in mammalian cells with functional approaches to gene discovery, suggested a potential alternative to complementation of mutants by using expression libraries in order to clone the missing gene. They indicated that the alternative involves retrovirus-mediated insertional mutagenesis in conjunction with chemical mutagenesis. They indicated that it was impractical to use insertional mutagenesis de novo to inactivate two alleles of a target gene. However, there was no indication of how this might be achieved and the reference failed to disclose a description of the method having been carried out in practice. It was suggested to obtain a population of heavily mutagenized cells and then insert retroviruses into those cells to inactivate and mark the gene. However, there was no report of this suggestion having been carried out, no guidance regarding how to perform either of the mutagenesis steps to achieve a successful homozygous mutation.
Accordingly, there exists a need in the art to create homozygous gene mutations on a genome-wide basis in cell culture and in multicellular organisms without knowledge of the gene in advance. There is also a need to provide a way to identify the gene. There is also a need for a method of mutating multiple genes in a cell, required cumulatively to achieve a desired phenotype and to identify one or more of the mutated genes. The ability to mutate multiple genes or to mutate both copies of the same gene in cultured cells or multi-cellular organisms, coupled with the ability to identify the mutant gene(s) would be a highly useful approach to identify novel genes, correlate genes with functions, and use the mutant genes, their wildtype counterparts, and other variants, for example, in drug screening and development, transgenic animal and plant production and in the production of desirable gene products.